Abstract
ZEB1 is a master regulator of the Epithelial-to-Mesenchymal Transition (EMT) program. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. In this work, we screened for potential regulatory links between ZEB1 and multiple cellular kinases. Exploratory in silico analysis aided by phospho-substrate antibodies and ZEB1 deletion mutants led us to identify several potential phospho-sites for the family of PKC kinases in the N-terminus of ZEB1. The analysis of breast cancer cell lines panels with different degrees of aggressiveness, together with the evaluation of a battery of kinase inhibitors, allowed us to expose a robust correlation between ZEB1 and PKCα both at mRNA and protein levels. Subsequent validation experiments using siRNAs against PKCα revealed that its knockdown leads to a concomitant decrease in ZEB1 levels, while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. Taken together, our findings unveil an unforeseen regulatory pathway comprising PKCα and ZEB1 that promotes the activation of the EMT in breast cancer cells.
Highlights
Epithelial-to-mesenchymal transition (EMT) is an essential program of normal embryonic development, tissue regeneration, organ fibrosis and wound healing [1, 2]
A positive correlation between ZEB1 and PKCα expression levels was found for the different breast cancer cell lines used in this analysis (r = 0.82, p < 0.001) (Figure 2D). Such correlation was not observed for other PKC isozymes known to have important roles in breast cancer progression, namely PKCδ and PKCε [45] (Supplementary Figures 2A,B). To expand on this finding, we bioinformatically explored the Heiser 2012 dataset (E-MTAB-181), where we cross-examined the mRNA levels of ZEB1 and PKCα in a larger panel of breast cancer cell lines
We focused on MDA-MB-231 breast cancer cells, a model that shows high levels of expression of ZEB1 and PKCα, and in which ZEB1 function has been extensively studied [48, 65, 66]
Summary
Epithelial-to-mesenchymal transition (EMT) is an essential program of normal embryonic development, tissue regeneration, organ fibrosis and wound healing [1, 2]. Activation of the EMT program is a critical step during metastatic expansion and for the generation of tumor cells with stem cell properties that play a major role in resistance to cancer treatment [3,4,5,6,7]. Several master regulatory programs have been discovered to play key roles in cancer progression and EMT, which can be activated by diverse signals, including TGF-β, Wnt, and TKR (tyrosine kinase receptor) ligands. These external signals regulate transcription factors (TFs) such as ZEB1, SNAIL, and TWIST by integrating molecular mechanisms which are still not fully understood [1, 3, 4, 8, 9]. The regulation and function of ZEB1 in a physiological context has been widely studied [19,20,21,22,23,24,25]
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